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Mass Spec Firm Spectroswiss Develops Tech to Enable Super Resolution on Orbitrap Instruments


NEW YORK – Mass spectrometry firm Spectroswiss has developed a signal processing approach and hardware that can boost the resolution of Thermo Fisher Scientific Orbitrap instruments.

The Lausanne, Switzerland-based company, a spinout from École Polytechnique Fédérale de Lausanne, has launched sales of the technology in the US and Europe. It is targeting it for several applications, including isobaric labeling-based proteomic workflows, said Yury Tsybin, the firm's founder and CEO.

Orbitraps are a type of Fourier transform instrument in which ions trapped in a chamber between an outer and an inner electrode oscillate with characteristic frequencies reflecting their mass-to-charge ratio. These frequencies are encoded into time-domain transients, which are converted into mass spectra using Fourier transforms, allowing researchers to identify the ion and measure its abundance.

The longer the duration of a transient, the higher the resolution of a measurement. Longer transients also mean slower mass spec scan speeds, however, which reduces the number of different ions that can be sampled in an experiment, negatively impacting sensitivity and depth of coverage. Spectroswiss' signal processing approach allows researchers to achieve higher resolution without sacrificing scan speed, Tsybin said, enabling super-resolution mass spectrometry.

Essentially, he said, the approach uses least-squares fitting to follow a narrow set of the possible frequencies corresponding to a particular transient, which allows researchers to boost the instrument's resolution without requiring longer transients.

"With the least-squares fitting, we say, 'We are looking for the particular and a priori known frequencies,'" Tsybin said. "We thus provide additional information that helps the method solve the problem."

Tsybin and his colleagues published a paper proposing their approach in 2014. Last month, they published a study in Analytical Chemistry demonstrating the use of the approach in an isobaric labeling workflow while showing a 20-fold increase in resolution.

There were several reasons it took nearly a decade to progress from the original 2014 paper to the recent demonstration of an application of the method, Tsybin said, one of the most significant being that Orbitrap instruments don't report the transient information required for the technique. This meant that Spectroswiss had to develop an instrument capable of interfacing with Orbitraps and recording and reporting transients. The company currently offers two systems for this purpose, the FTMS Booster X2 and the FTMS Booster X2T.

Identifying applications well suited to the approach was also key, Tsybin said. "Once we developed the devices to collect transients from Orbitraps, we needed to find biological problems and methods where the [method] would shine."

The company looked at isobaric labeling, for example, first exploring conventional tandem mass tag (TMT) labeling where Tsybin and his colleagues thought a boost in resolving power could allow for increased multiplexing or could speed analyses at existing levels of multiplexing.

Isobaric labeling uses stable isotope tags attached to peptides of interest to enable relative or absolute quantitation of proteins via tandem mass spectrometry. Digested peptides are labeled with tags that fragment during MS2 to produce reporter ions corresponding to the amount of peptide present in a sample. The reporter ions produced by each set of tags differ slightly in mass from the others, allowing researchers to track which sample produced which tags. The approach is commonly used to multiplex samples, with TMT technology currently enabling multiplexing of up to 18 samples. Increased multiplexing, however, means that the reporter ions are separated by smaller amounts of mass, which, in turn, requires higher instrument resolving power or longer cycle times.

Looking into the application of Spectroswiss' technology to TMT, the company determined that while there was a benefit, it wasn't substantial enough to make for a solid market opportunity for a small company, Tsybin said.

Additionally, Thermo Fisher had developed an in-house super-resolution approach called Phi-SDM that it applied to TMT multiplexing in the TurboTMT offering on its Orbitrap Exploris 480. The approach, which the company has said boosts resolution by twofold to threefold at any transient length, allows for faster scan rates and increased proteome coverage in TMT experiments.

Tsybin saw potential, though, for Spectroswiss' super-resolution technology in another TMT workflow, a method called TMTc that was codeveloped by the lab of Princeton University researcher Martin Wühr and aims to address the problem of precursor interference in isobaric labeling.

Precursor interference happens when non-target ions slip through in the experiment. Because these ions have also been labeled with isobaric tags, they contribute to the reporter signal for the target peptide, which decreases the accuracy with which the target is measured.

The TMTc approach tackles this problem by measuring not the reporter ions but rather the peptides plus the portion of the reporter tag left attached to the peptide after fragmentation. This allows researchers to retain the distinct isobaric tag information that allows multiplexing of different samples, while also measuring the peptide it was attached to, resulting in reporter ions, termed TMTc ions, with different masses for each peptide. The approach lets researchers distinguish between reporter ions even in the case of co-isolating peptides.

The large mass of the TMTc reporters as opposed to traditional TMT reporters presents a challenge, however, in that instrument resolution drops at high mass ranges, Tsybin said.

"You need super resolution or else the lengths of the transients [required] will be prohibitive," he said, suggesting as a target a 50-fold resolution increase.

He noted that he and his colleagues did not manage a 50-fold increase in resolution in their recent Analytical Chemistry paper but did achieve a 20-fold increase.

"That brings us into the range of scanning speed needed for TMTc in proteomics," he said.

Wühr, who was a coauthor on the Analytical Chemistry paper, said that the resolution challenges involved in TMTc had thus far limited the approach to nine-plex experiments, as opposed to the 18-plex experiments possible with conventional TMT labeling.

"This seemed like a great opportunity to fix that and being able to use more channels with these super-resolution approaches," he said. "I believe the paper has provided a nice proof of principle that this is viable."

Wühr said his lab plans to continue evaluating the approach. He added that perhaps the biggest hurdle facing Spectroswiss' super-resolution technology is streamlining the data analysis involved.

The super-resolution method "seems to work pretty well, but the software is all a little bit still a prototype, and Yury and his team are working on streamlining it so that we can really regularly push through our biological samples with this kind of approach," he said.

Tsybin cited single-cell proteomics as another application where the technique could prove advantageous. Isobaric labeling is commonly used in single-cell proteomic workflows, and some researchers — including at Thermo Fisher — have explored super resolution combined with TMTc for single-cell experiments.

An additional application might be mass spec imaging, he added.

Tsybin noted that while Thermo Fisher is also pursuing super-resolution approaches, Spectroswiss, as a smaller company, might have the flexibility to address applications that might be too narrow for the life science giant to go after.

"It will be a matter of who will have the better performance, and also what the application areas will be, because Thermo is not working on all application areas, and for us even niche application areas can be attractive," he said.

Bernhard Küster, chair of proteomics and bioanalytics at the Technical University of Munich, said that he does not see the approach as a "game changer" and that its impact on proteomics remains unclear to him. Küster, who was not involved in the work, said he agrees, though, with the authors of the Analytical Chemistry paper that it is "a welcome addition to the arsenal" of mass spectrometry methods.